Establishment of a versatile histidine-rich glycoprotein (HRG) measurement methodology bearing a highly compatible design for patients with sepsis in daily clinical practice

Sepsis is a life-threatening condition that requires reliable biomarkers for appropriate clinical assessment. Histidine-rich glycoprotein (HRG) has emerged as a promising biomarker candidate associated with sepsis severity. We previously developed an ELISA method using monoclonal antibodies, 69-1 A and 75-2D for sensitive and specific quantification of plasma HRG. However, the assay required extensive sample dilution and further optimization to improve analytical robustness. In the present study, we developed an optimized ELISA by reducing sample dilution ratio and extending the measurable range toward higher HRG concentrations through a competitive reaction system incorporating a small amount of free 75-2D antibody. The optimized assay covered a plasma HRG range of 16-250 μg/mL and showed lower coefficients of variation than the previous method. The optimized method also showed good correlation with the previous ELISA. In addition, we evaluated recombinant single-isoform HRG protein as candidate calibrator material for this assay system. Because both isoforms showed comparable reactivity and dilution linearity, recombinant Form-1 HRG (an isoform bearing Pro at 204-position, 75 kDa) was selected as a practical candidate calibrator based on its simpler glycosylation-related characteristics and quantitative assignment by amino acid analysis. Our new findings support the optimization of ELISA system and a single-isoform recombinant calibrator for more reproducible HRG measurement. It is expected that analytical standardization and clinical validation will subsequently be performed to establish in vitro diagnostic application in actual clinical use.